Determination of the presence or absence of a functional genetic segment in a sample or individual is of great importance in several fields, from forensics, to systematics, to diagnostics and health management. For example, deletions involving tumor suppressor genes are believed to play an important role in the development of several types of cancer (Dutrillaux et al., 1990; Hensel et al., 1990). In the case of human blood groups, absence of certain segments has great implications for blood group phenotypes, which in turn has an effect on transfusion compatibility. For example, Human RhDel is caused by a deletion of 1,013 bp which includes exon 9 of the RHD gene (Chang et al. 1998).
A number of methods are known and used to assess the presence or absence of specific genetic segments. These include sequencing—(Sanger and Coulson, 1975; Smith et al., 1986) based approaches (Kitano et al., 2000) as well as the use of electrophore or Southern blotting (Southern, 1975) to detect markers such as restriction fragment length polymorphisms (RFLP, Botstein et al., 1980) or other markers based on the polymerase chain reaction (PCR, Mullis et al., 1986; Mullis and Faloona, 1987), such as amplicons from sequence-specific primers (Olerup and Zetterquist, 1992) or single-stranded conformational polymorphisms of PCR products (Jin et al. 1993).
Several patents and/or patent applications describe as well methods for detecting absence or deletions of certain functional genetic segments, or their use for certain objectives. For instance, patent application WO 2010/008071 discloses a method for screening the presence or absence of an exon having any gene mutation therein. The method is a gene mutation screening method utilizing a quantitative PCR technique. U.S. Pat. No. 6,599,701 provides methods for characterizing organisms by identifying the presence, absence, size, or sequence polymorphism of intronic regions. WO 2001/018245 provides methods of identifying an alteration in a gene of interest, particularly in the major histocompatibility region, utilizing long range polymerase chain reaction (LR-PCR) amplification of target DNA that includes all or a portion of a human mobile element.
The present invention aims at providing methods for the selection and use of probe sets that allow detecting the presence or absence of functional segments and also in certain cases, both to detect allelic variants located within their cognate sequences and to determine the presence or absence of functional segments simultaneously, in a high-through put way.